Project Summary/Abstract Rett syndrome (RTT), a devastating neurodevelopmental disorder, is largely caused by loss-of-function mutations in the X-linked gene encoding the epigenetic regulator MeCP2. Our group was the first to show a detrimental role of microglial abnormalities in a MECP2 knockout (MECP2-KO) mouse model of RTT. Recent in vivo data suggest that correcting microglial abnormalities is sufficient to rectify major RTT-like symptoms in this model. Therefore, how MeCP2 deficiency causes microglial abnormalities and how the functional states of MeCP2-deficient microglia (RTT-MG) influence RTT pathology is a critical question. Recently we found that MeCP2 deficiency in RTT-MG resulted in enhanced mitochondrial reactive oxygen species (mtROS) production, which we hypothesize would lead to metabolic derangements and abnormal microglial functions. Interestingly, in vitro studies showed that several key RTT-MG abnormalities were rescued by mitochondria- targeted transgene mCAT (the antioxidant enzyme catalase being expressed only in mitochondria, which usually do not harbor catalase) and two FDA-approved mitoactive Nrf2 activators, opening a possibility for intervention. Our preliminary data further showed that global expression of mCAT (GL-mCAT) prolonged the lifespan and improved motor and respiratory functions of the MECP2-KO mice, supporting the promise of our approach in a whole animal setting. Here we propose to extend this line of research to gain a deeper understanding of how MeCP2 is related to functional and pathological states of microglia, and to generate preclinical proof of principle that is instrumental for developing novel therapies for RTT: Aim 1: Determine the pathological role of microglial mtROS in vivo: Several lines of evidence support that microglia abnormalities drive the progression of RTT. We have shown that global expression of mCAT ameliorates the RTT-like phenotype in MECP2-KO mice. Based on this encouraging result, we further hypothesize that quenching mtROS selectively in microglia to rectify microglial abnormalities will also ameliorate RTT-like deficits in MECP2-KO mice. For this aim, we will generate and analyze the phenotype and microglial pathology of MECP2-KO/MG-mCAT mice with microglia-targeted expression of mCAT. The result would support the role of microglial mtROS in the pathogenesis of RTT. Aim 2: Determine the role of mtROS in pathological characteristics and functional responsiveness of RTT microglia: Our previous data, mostly in vitro work, support the hypothesis that mtROS-related metabolic/molecular derangements lead to RTT-MG abnormalities including the loss of the ?metabolic flexibility? required to drive their functional responsiveness. Now in this aim we will test this hypothesis in vivo, mainly by analyzing microglia acutely isolated from MECP2-KO and MECP2-KO/mCAT mice. We will determine if mCAT, expressed in vivo, is able to (a) rectify the metabolic/molecular derangements of RTT-MG, and (b) recover the ability of RTT-MG to respond to M1/M2-inducing signals with proper functional differentiation. Key microglial cellular features such as phagocytic function, mitochondrial integrity, aerobic glycolysis, functional polarization, and Nrf2 antioxidation pathway will be analyzed to determine the beneficial effects of mtROS suppression by mCAT. Aim 3: Test the therapeutic effects of two FDA-approved mitoactive/Nrf2 activating drugs in vivo: The success of the mCAT approach provides a rationale for anti-mtROS therapy. To translate this finding to potential therapies, we screened 1,600 FDA-approved drugs to identify mitoactive anti-mtROS compounds, and subsequently screened the hits on a RTT-MG culture platform. We now have two leads able to correct RTT- MG abnormalities in vitro. Interestingly, both are known activators of the Nrf2 antioxidation pathway, supporting our hypothesized pivotal role of Nrf2 in RTT-MG. Because of their known favorable pharmacokinetics/safety profiles, now we will test them in vivo; efficacy shown in this aim would enable rapid translation by re-purposing these FDA-approved drugs for RTT. In summary, these studies will clarify mechanisms of MeCP2-regulated microglial function, the role of microglial mtROS in RTT, and advance novel therapies for RTT, for which no effective treatment is available. We hope that findings in this proposal would set the foundation to explore several new areas, such as how mtROS and metabolic modules such as aerobic glycolysis mediate or modulate microglial function including M1/M2 differentiation and phagocytosis, and the role of Nrf2 in microglial function. Moreover, RTT is one of few Autism Spectrum Disorders (ASDs) whose cause is identified as a single gene mutation and shares important pathogenic pathways with autism. Our studies, therefore, may implicate novel mechanisms and therapeutic approaches as well for autism where mitochondrial and microglia dysfunction could also play a role.